Integrating mRNA and Protein Sequencing Enables the Detection and Quantitative Profiling of Natural Protein Sequence Variants of <i>Populus trichocarpa</i>

Next-generation sequencing has transformed the ability to link genotypes to phenotypes and facilitates the dissection of genetic contribution to complex traits. However, it is challenging to link genetic variants with the perturbed functional effects on proteins encoded by such genes. Here we show how RNA sequencing can be exploited to construct genotype-specific protein sequence databases to assess natural variation in proteins, providing information about the molecular toolbox driving cellular processes. For this study, we used two natural genotypes selected from a recent genome-wide association study of <i>Populus trichocarpa</i>, an obligate outcrosser with tremendous phenotypic variation across the natural population. This strategy allowed us to comprehensively catalogue proteins containing single amino acid polymorphisms (SAAPs), as well as insertions and deletions. We profiled the frequency of 128 types of naturally occurring amino acid substitutions, including both expected (neutral) and unexpected (non-neutral) SAAPs, with a subset occurring in regions of the genome having strong polymorphism patterns consistent with recent positive and/or divergent selection. By zeroing in on the molecular signatures of these important regions that might have previously been uncharacterized, we now provide a high-resolution molecular inventory that should improve accessibility and subsequent identification of natural protein variants in future genotype-to-phenotype studies

AMMI analysis results for location specificity in QTL detection between the Clatskanie and Boardman sites.

<p>AMMI analysis results for location specificity in QTL detection between the Clatskanie and Boardman sites.</p

Suggestive QTLs associated with height and diameter identified in <i>Populus</i> family 331 F₂ pedigree.

†<p>LG = Linkage groupwise significance; GW = Genomewise significance.</p

Genome Anchored QTLs for Biomass Productivity in Hybrid <em>Populus</em> Grown under Contrasting Environments

<div><p>Traits related to biomass production were analyzed for the presence of quantitative trait loci (QTLs) in a <em>Populus trichocarpa</em> × <em>P. deltoides</em> F₂ population. A genetic linkage map composed of 841 SSR, AFLP, and RAPD markers and phenotypic data from 310 progeny were used to identify genomic regions harboring biomass QTLs. Twelve intervals were identified, of which <em>BM-1</em>, <em>BM-2</em>, and <em>BM-7</em> were identified in all three years for both height and diameter. One putative QTL, <em>BM-7,</em> and one suggestive QTL exhibited significant evidence of over-dominance in all three years for both traits. Conversely, QTLs <em>BM-4</em> and <em>BM-6</em> exhibited evidence of under-dominance in both environments for height and diameter. Seven of the nine QTLs were successfully anchored, and QTL peak positions were estimated for each one on the <em>P. trichocarpa</em> genome assembly using flanking SSR markers with known physical positions. Of the 3,031 genes located in genome-anchored QTL intervals, 1,892 had PFAM annotations. Of these, 1,313, representing 255 unique annotations, had at least one duplicate copy in a QTL interval identified on a separate scaffold. This observation suggests that some QTLs identified in this study may have shared the same ancestral sequence prior to the salicoid genome duplication in <em>Populus</em>.</p> </div

LOD traces for QTL B<i>M-2</i> on LG II based on 4-year height (red) and 4-year diameter (green) measured in Clatskanie and 4-year height measured in Boardman (purple).

<p>Broken horizontal line represents linkage groupwise LOD significance threshold calculated based on 1,000 permutations at the 0.05 significance level.</p

QTLs associated with height and diameter identified in <i>Populus</i> family 331 F₂ pedigree based on linkage-group- (LG) and genome-wise LOD significance thresholds (GW).

<p>%PVE = percent phenotypic variance explained;</p>†<p>Mean associated with heterozygous genotypes ‘ac’ and ‘bd’ where alleles are derived from the same species;</p>‡<p>Mean associated with heterozygous genotypes ‘ad’ and ‘bc’ where alleles are derived from different species, a = additive; d = dominance; d/|a| = QTL mode of action.</p

Synteny between (A) Family 331 genetic map LG II, (B) <i>Populus</i> consensus genetic map LG II, and (C) Scaffold 2 of the <i>Populus</i> genome assembly illustrating the genome anchoring of QTL <i>BM-2</i> using flanking markers.

<p>Map distance units in A and B represent cM distances and distance units in C represent genomic sequence length (x1OKb).</p

Genome anchored QTL positions on the <i>Populus</i> V2.2 assembly.

<p>Blue bars represent SSR marker coverage for each scaffold, red bars indicate scaffold intervals between flanking SSR markers used for genome anchoring, vertical green lines represent QTL intervals and estimated peak position. Scaffold intervals are represented in Mb.</p

Defining the Boundaries and Characterizing the Landscape of Functional Genome Expression in Vascular Tissues of <i>Populus</i> using Shotgun Proteomics

Current state-of-the-art experimental and computational proteomic approaches were integrated to obtain a comprehensive protein profile of <i>Populus</i> vascular tissue. This featured: (1) a large sample set consisting of two genotypes grown under normal and tension stress conditions, (2) bioinformatics clustering to effectively handle gene duplication, and (3) an informatics approach to track and identify single amino acid polymorphisms (SAAPs). By applying a clustering algorithm to the <i>Populus</i> database, the number of protein entries decreased from 64689 <i>proteins</i> to a total of 43069 <i>protein groups</i>, thereby reducing 7505 identified proteins to a total of 4226 protein groups, in which 2016 were singletons. This reduction implies that ∼50% of the measured proteins shared extensive sequence homology. Using conservative search criteria, we were able to identify 1354 peptides containing a SAAP and 201 peptides that become tryptic due to a K or R substitution. These newly identified peptides correspond to 502 proteins, including 97 previously unidentified proteins. In total, the integration of deep proteome measurements on an extensive sample set with protein clustering and peptide sequence variants provided an exceptional level of proteome characterization for <i>Populus</i>, allowing us to spatially resolve the vascular tissue proteome

Supplemental Material for Bryan et al., 2018

Supplementary data of "A variable polyglutamine repeat affects subcellular localization and regulatory activity of a <i>Populus</i> ANGUSTIFOLIA protein